Ectopic Expression of a <i>Glycine soja myo</i>-Inositol Oxygenase Gene (<i>GsMIOX1a</i>) in <i>Arabidopsis</i> Enhances Tolerance to Alkaline Stress

<div><p><i>Myo</i>-inositol participates in various aspects of plant physiology, and <i>myo</i>-inositol oxygenase is the key enzyme of the <i>myo</i>-inositol oxygenation pathway. Previous studies indicated that <i>myo</i>-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of <i>GsMIOX1a</i>, a remarkable alkaline stress-responsive gene of <i>Glycine soja</i> 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that <i>GsMIOX1a</i> is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of <i>GsMIOX1a</i> in the alkaline response in the wild type, <i>atmiox1</i> mutant as well as <i>GsMIOX1a</i>-overexpressing <i>Arabidopsis</i>. We determined that <i>atmiox1</i> mutant decreased <i>Arabidopsis</i> tolerance to alkaline stress, whereas <i>GsMIOX1a</i> overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including <i>H⁺-Ppase, NADP-ME, KIN1</i> and <i>RD29B</i>, were also up-regulated in <i>GsMIOX1a</i> overexpression lines compared with the wild type and <i>atmiox1</i> mutant. Together, these results suggest that the <i>GsMIOX1a</i> gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of <i>myo</i>-inositol oxygenase improves alkaline tolerance in plants.</p></div

The phylogenetic tree of GsMIOX1a and MIOX from different plants.

<p>The analysis was based on a multiple amino acid sequence alignment. A neighbor-joining tree was generated using MEGA 5.0. The aligned protein sequences from GenBank (with accession numbers) included GsMIOX1 (<i>Glycine soja</i>, KHN39268), AtMIOX1 (<i>Arabidopsis thaliana</i>, NP_001154337), AtMIOX2 (<i>Arabidopsis thaliana</i>, NP_565459), AtMIOX4 (<i>Arabidopsis thaliana</i>, NP_194356), AtMIOX5 (<i>Arabidopsis thaliana</i>, NP_200475), MtMIOX (<i>Medicago truncatula</i>, KEH18252), CaMIOX (<i>Cicer arietinum</i>, XP_004510326), OsMIOX (<i>Oryza sativa</i>, NP_001057871), ZmMIOX (<i>Zea mays</i>, NP_001141330), PvMIOX (<i>Phaseolus vulgaris</i>, XP_007135612), GaMIOX (<i>Gossypium arboretum</i>, KHF98793), SiMIOX (<i>Setaria italica</i>, XP_004966088), GmMIOX1a (<i>Glycine max</i>, Glyma07g01660), GmMIOX1b (<i>Glycine max</i>, Glyma08g21300), GmMIOX2a (<i>Glycine max</i>, Glyma01g00840), GmMIOX2b (<i>Glycine max</i>, Glyma07g15190), GmMIOX4 (<i>Glycine max</i>, Glyma08g01690), SbMIOX (<i>Sorghum bicolor</i>, Sb10g022160), and RcMIOX (<i>Ricinus communis</i>, 29912.m005347) with gene ID from Phytozome v9.1.</p

Effect of alkaline stress on mature plants of the WT, <i>atmiox1</i>, and <i>GsMIOX1a</i> OX lines.

<p>(A) Phenotypes of the WT, <i>atmiox1</i> and OX plants in response to 100 mM NaHCO₃ stress. Three-week-old <i>Arabidopsis</i> plants were irrigated with 100 mM NaHCO₃ solution every 3 days for a total of 12 days. The photographs were taken on the 13th day after stress exposure. (B) The free proline content of the WT, <i>atmiox1</i> and OX plants under control conditions or stress treatment. (C) The POD activity of the WT, <i>atmiox1</i> and OX plants under control conditions or alkaline stress. (D-E) The AsA content in the leaf extracts of the <i>GsMIOX1a</i> OX <i>Arabidopsis</i>, <i>atmiox1</i> and WT lines. The total amount of AsA comprised the reduced (AsA) and oxidized forms (DHA). AsA contents were measured in the leaves of 5-week-old <i>GsMIOX1a</i> OX <i>Arabidopsis</i>, <i>atmiox1</i> and WT plants under normal and alkaline stress conditions in a growth chamber. Leaves of the same age and size were selected to minimize experimental error. Error bars represent the ± S.E. (n = 3). Significant differences were analyses using the T-test method. Significant differences from WT are denoted by one or two stars, corresponding to p<0.05 and p<0.01, respectively.</p

Basic information on the MIOX family in <i>Arabidopsis thaliana</i> and <i>G</i>. <i>max</i>.

<p>Basic information on the MIOX family in <i>Arabidopsis thaliana</i> and <i>G</i>. <i>max</i>.</p

Expression of marker genes in the WT, <i>atmiox1</i> and <i>GsMIOX1a</i> OX seedlings under alkaline stress.

<p>(A) <i>H</i>^<i>+</i>-<i>Ppase</i> expression levels under alkaline stress. (B) <i>KIN1</i> expression levels under alkaline stress. (C) <i>NADP-ME</i> expression levels under alkaline stress. (D) <i>RD29B</i> expression levels under alkaline stress. To explore the transcript levels of stress-responsive genes, 2-week-old WT, <i>atmiox1</i> and OX seedlings (line #4) were treated with 1/2 MS solution containing 50 mM NaHCO₃ (pH 8.5) for 0, 1, 3, 6, and 12 h. The relative transcript levels were determined by quantitative real-time PCR. The <i>AtActin2</i> gene served as an internal reference, and transcript levels were normalized to WT at 0 h. The values represent the means of three independent biological replicates and three technological replicates for each. One or two stars correspond to p<0.05 and p<0.01, respectively.</p

Additional file 2: Figure S1.

Nucleotide sequence and deduced amino acid sequence of BpMYC4 from Betula platyphylla. (DOCX 287 kb

Additional file 5: Figures S5, S6 and S7.

Identification and acquisition of BpbHLH9 transgenic seedlings. (DOCX 730 kb

Additional file 3: Figure S2.

Nucleotide sequence and deduced amino acid sequence of BpbHLH9 from Betula platyphylla. (DOCX 193 kb

Additional file 1: Table S1. of Cloning and expression of BpMYC4 and BpbHLH9 genes and the role of BpbHLH9 in triterpenoid synthesis in birch

List of primers used in the study. (DOCX 16 kb

Ectopic expression of <i>GsCML27</i> in <i>Arabidopsis</i> influences plant by modifying cellular ionic content.

<p><b>A</b>. Growth performance of WT and ectopic expression seedlings on 1/2 MS medium with 50, 100, 150 mM KCl. Seedlings with open and green leaves was recorded 7 days after stratification. <b>B</b>. Growth performance of WT and ectopic expression seedlings on 1/2 MS medium with 10, 20, 30 mM LiCl. Seedlings with open and green leaves was recorded 7 days after stratification. Data shown represent the means (±SE) of three independent experiments. *P < 0.05 and **P < 0.01 by Student’s t-test.</p